Preparation of purified (poly)peptides

ABSTRACT

The invention provides for a method for the modification of (poly)peptides for facilitating purification thereof, which modification method comprises the insertion of at least one specifically cleavable amino acid at the end of the (poly)peptide chain during synthesis thereof and protecting the same amino acid(s) within the (poly)peptide, if present, against cleavage, in order to allow for specific cleavage precisely at the specifically cleavable amino acid(s). The invention further relates to a process for the preparation of purified (poly)peptides using the modification method. The process comprises the steps of synthesizing the desired (poly)peptide; adding at least one specifically cleavable amino acid at the end of the (poly)peptide, while having protected the same amino acid(s) within the (poly)peptide, if present, against cleavage; elongating the (poly)peptide and the amino acid(s) added thereto with a tag sequence to obtain an elongated (poly)peptide; purifying the elongated (poly)peptide by means of a tag-specific purification method; and removing the tag sequence and the additional amino acid(s) from the elongated (poly)peptide by means of a cleavage method specific for the additional amino acid(s) to obtain the purified (poly)peptide.

This is a U.S. National Stage Application of PCT/EP97/04939 filed Sep.9, 1997.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a modification method for facilitatingthe preparation of purified (poly)peptides and to a purification processusing this modification method.

2. Description of the Related Art

During the last years solid phase peptide synthesis, using either t-Bocor F-moc strategies, has been largely improved. Sophisticated protocolsof synthesis allowed the preparation of polypeptides of about 100residues or more¹⁻⁴. Nevertheless, incomplete coupling and chaintermination that may occur at any cycle of the peptide assembly leads tothe formation of deletion--and truncated sequences.

This and the possible occurrence of side reactions observed mainlyduring the final cleavage from the resin hamper the straightforwardisolation of the desired peptide from other impurities. The purificationof long synthetic polypeptides is a major problem in the production ofproducts useful for biological studies and for human and animal usewhere a high level of purity is mandatory.

In particular, when sequences containing 30 or more residues aresynthesized, the differences in physical properties such as size, chargeand hydrophobicity between the desired product and deleted, truncated ormodified peptide impurities may be too small to allow adequatepurification. In addition, the modern powerful separation techniques,i.e. reverse phase HPLC, are often limited by low yields and smallsample loadability which is time-consuming and expensive. Differentapproaches have been already tested to circumvent this limitation.Biotinylation of a 153 residue IL-1 synthetic protein⁵ and of a 99residue SIV protease synthetic protein⁶ were performed and thebiotinylated chains were isolated on an avidin-agarose column.

Ball et al.⁷ have recently proposed a purification procedure based onthe addition of a reversible protecting group which bears eitherlipophilic, acidic or basic functions to the last residue of the peptidechain.

More specific chromatographic methods have been optimized exploiting thepresence of particular residues in the synthesized sequences. Forexample, cysteine-containing peptides have been purified by reactionwith immobilized mercury derivatives⁸ or activated thiols⁹ andimmobilized metal ion affinity chromatography (IMAC)¹⁰ has beensuccessfully applied for the purification of peptides containinghistidine or tryptophan¹¹.

In recombinant proteins a histidine tail, B cell epitope or GST moietieswere purposely added. These tails could subsequently be used in affinitychromatography.

In general, a purification protocol which is based on thephysico-chemical properties of the synthesized peptide has to beoptimized for each individual sample, which is a time-consuming andcostly exercise. For these reasons, a number of techniques have beendeveloped to render purification procedures of general applicability¹⁵.However, the methods described so far are not completely satisfactorysince they are still time consuming and/or leave covalently derivatizedpeptides in the final purified products which may pose some concern fortheir biological and physico-chemical properties and their finalutilization in animals and humans.

It is therefore the object of the present invention to provide animprovement to the known methods by providing a method for themodification of (poly)peptides for facilitating purification thereof,and to a purification process for (poly)peptides, which method andprocess are universally applicable, result in a high yield of recoveryand are easy to perform.

This object was achieved according to the invention by a modificationmethod, which comprises the insertion of at least one specificallycleavable amino acid at the end of the (poly)peptide chain duringsynthesis thereof and protecting the same amino acid(s) within the(poly)peptide, if present, against cleavage, in order to allow forspecific cleavage precisely at the specifically cleavable amino acid(s).

SUMMARY OF THE INVENTION

A purification process using this modification method, comprises thesteps of:

a) synthesizing the desired (poly)peptide;

b) adding at least one specifically cleavable amino acid at the end ofthe (poly)peptide, while having protected the same amino acid(s) withinthe (poly)-peptide, if present, against cleavage;

b) elongating the (poly)peptide and the amino acid(s) added thereto witha tag sequence to obtain an elongated polypeptide;

c) purifying the elongated polypeptide by means of a tag-specificpurification method; and

d) removing the tag sequence and the additional amino acid(s) from theelongated (poly)peptide by means of a cleavage method specific for theadditional amino acid(s) to obtain the purified polypeptide.

The specific cleavage method is preferably a chemical cleavage method aswill be further elucidated hereinbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a mass spectrum of crude peptide;

FIG. 2 is a chromatographic profile of IMAC purification;

FIG. 3 is a mass spectrum of purified peptide; and

FIGS. 4A and 4B are chromatographic profiles of crude and purifiedpeptide.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The method and process of the invention are applicable to every(poly)peptide since they do not depend on the amino acid compositionthereof. Furthermore, in certain preferred embodiments the method andprocess are inexpensive and highly efficient.

Such an embodiment is the use of a methionine residue as the additionalamino acid before adding the affinity tag (for example a stretch of sixhistidine residues or other purification facilitating compounds). Afterappropriate purification steps, such as tag-specific affinitychromatography, the histidine tag may be cleaved of by CNBr digestion,an inexpensive and very efficient process, which specifically cleaves atthe methionine residue.

In a preferred embodiment the tag thus comprises at least a stretch ofhistidine residues, preferably six or more, and a methionine residue.Optionally one or more other amino acids may be incorporated.

If the sequence of the desired polypeptide contains methionine residuesthey would be subject to cleavage by cyanogen bromide when removing thetag. However, according to the invention this may be avoided by usingmodified methionine residues in the synthesis of the (poly)peptide. Sucha modified methionine residue is for example methionine sulfoxide.

In an alternative embodiment of the method and process of the inventionthe tag is a large molecule, such as polyethylene glycol. In this casethe tag-specific purification method is gel filtration chromatography.

The method and process of the invention are equally well applicable topolypeptides produced by recombinant DNA techniques. In a preferredembodiment the methionine residues originally present within the desiredpolypeptide, but not in the tag are cleavage protected, for example bybeing substituted by another amino acid, such as valine, glycine, ordeleted.

In this application "tag" is used to indicate a removable molecule addedto the desired polypeptide during or after synthesis thereof. A "tag"may be an amino acid sequence added during synthesis of the polypeptide,but may also be another molecule than can be easily purified out of amixture of components. An example of the latter is polyethylene glycol(PEG).

In this application the terms "peptide", "polypeptide" and"(poly)peptide" are used interchangeably.

The next example is given for illustrating the invention. It is clearthat for the skilled person this example will give enough guidance todevelop further methods that fall within the scope of the invention. Inthe example a purification procedure of general applicability isdisclosed, based on the combination of 1) a capping protocol, 2) use ofmethionine sulfoxide as protected methionine residue during assembly ofthe native sequence and 3) elongation of the desired peptide with amethionine and 2 glycine residues and a final stretch of 6 histidineswhich will be used for chromatography affinity. After adequatepurification steps, cyanogen bromide cleavage of the histidine tagfollowed by final reduction of methionine sulfoxide to methionine wasperformed. This simple, straightforward strategy allowed thepurification to homogeneity of the 69 residue polypeptide "PbCS242-310", covering the C-terminal region of the Plasmodium berghei CSprotein, in high yield and short time using conventional chromatographicprocedures.

EXAMPLE

1. Materials and Methods

1:1. Reagents and Solvents

Chemicals and solvents used for the peptide synthesis were purchasedfrom Calbiochem-Novabiochem AG (Laufelfingen, Switzerland) and Fluka(Buchs, Switzerland).

1.2. Peptide Synthesis and Analysis

To illustrate the present invention a polypeptide designated "PbCS242-310" covering the C-terminal region of Plasmodium berghei CSprotein¹² was chemically synthesized using solid phase F-Moc chemistryin an Applied Biosystems 431A Peptide Synthesiser. The polypeptide wasprepared on a F-moc-Ser(t-butyl)-p-alkoxybenzylalkohol resin (Wangresin) with a degree of substitution of 0.43 mmol/g at the 0.1 mmolscale. The synthesis was performed using a five fold excess of F-mocamino acid derivatives, DCCI and HOBt as activating agents, a 60 mincoupling time for the first 34 amino acids and a double coupling for thefollowing residues. Capping with acetic anhydride was performed at theend of each cycle. Side chain protecting groups included:pentamethylchroman sulfonyl group for Arg; -S-t-butyl for Cys;triphenylmethyl group for Asn, Gln and His; t-butoxycarbonyl group forLys and Trp; t-butyl group for Asp, Clu, Ser, Thr and Tyr. Met 306 wasinserted as Fmoc-Met-sulfoxide to protect it against later cyanogenbromide cleavage.

The peptide was then elongated N-terminally with the sequenceHis-His-His-His-His-His-Gly-Gly-Met (SEQ ID NO: 1) using the sameconditions described above but capping was omitted after coupling of thesecond Gly. The polypeptide thus obtained is designated "His tag PbCS242-310".

Crude peptide was obtained by treating the peptide-resin with 2.5% H₂ O,5% triethylsilan in TFA for 2 hours at room temperature. Syntheticpeptide was purified by size exclusion liquid chromatography (SephadexG50 column 70×2.5 cm using 50% acetic acid/H₂ O as mobile phase). Thepurity of peptide was analyzed by RP-HPLC using a C4 W-Porex 250×4.6 mmcolumn and a 10-90% CH₃ CN gradient in 0.1% TFA/H₂ O in 60 min with aflow rate of 1.0 ml/min. The amino acid composition was determinedaccording to Knecht and Chang¹³.

1.3. Immobilized Metal Ion Affinity Chromatography (IMAC) and CNBrCleavage

The polypeptide was first purified by affinity chromatography based onthe histidine tag. Afterwards the tag was removed by cyanogen bromide.

A Ni-column was prepared with Ni-NTA agarose resin (Qiagen Inc.,Chatsworth, USA) and equilibrated with buffer A (8M urea, 0.1 M Na₂HPO₄, 0.01 M Tris, pH adjusted to 8.0 with H₃ PO₄). Size exclusionpurified "His-tag PbCS 242-310" polypeptide was dissolved in Buffer Aand loaded on the column with a flow rate of 15 ml/h. The column waswashed with buffer A (flow rate 15 ml/h) and buffer B (8M urea, 0.1 MNa₂ HPO₄, 0.01 M Tris, pH adjusted to 6.3 with H₃ PO₄) containing 50 mMimidazole at a flow rate of 30 ml/h. The "His-tag PbCS 242-310"polypeptide was then eluted (flow rate 30 ml/h) with buffer B containing250 mM imidazole.

The eluted material was desalted by a Sephadex G25 column (50×2.5 cmusing 50% acetic acid/H₂ O as mobile phase) and lyophilized. For removalof the histidine tag the thus obtained material was treated for 8 hoursat RT at a concentration of 20 mg/ml in 70% TFA using a 100 fold molarexcess of CNBr.

The thus digested material was lyophilized, solubilized in Buffer A andloaded again on the Ni-NTA agarose column. The histidine tag.is retainedon the Ni-column and the flow through of the column contains the "PbCS242-310" polypeptide. The flow through was desalted by a Sephadex G25column (50×2.5 cm using 50% acetic acid/H₂ O as mobile phase) andlyophilized.

1.4. Met-Sulfoxide Reduction

The CnBr treated and IMAC purified material was treated with 10%mercaptoethanol at pH 8.0 to convert methionine sulfoxide intomethionine and Cys-S-t-butyl into Cys and then further purified by gelfiltration (Sephadex G25 column 250×4.4 mm).

1.5. Mass Spectrometry

Mass Spectrometry analysis was performed using a time-of-flight massspectrometer LDI 1700 Mass Monitor (Linear Scientific Inc., Reno, N.V.,USA). Five μl of a solution of 1 mg/ml of polypeptide were mixed with 5μl of trans-3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid) (20mg/ml in acetonitrile (Linear Scientific Inc. Reno, N.V., USA)) and 1.0μl of this solution was placed on the mass spectrometer probe tip anddried with a gently vacuum. The sample was irradiated with 3-ns laserpulses (wavelength 337 nm) from a N₂ -Laser. Time-of-flight was measuredwith a digital oscilloscope (series 9304; Le Croy Research Systems,Corp., Spring Valley, N.Y.) which was converted into mass spectrum usingthe Peptide MALDI-TOFMS Calibration Standard (Linear Scientific Inc.,Reno, N.V., USA).

2. Results

The 69 residue polypeptide "PbCS 242-310" corresponds to the C-terminalregion of P. berghei CS protein¹². Synthesis of "His-tag PbCS 242-310"was performed using an automatic protocol in which a capping step wasincluded after each coupling as described in the "Materials and Methods"section.

More than 150 mg of crude polypeptide were obtained by treating 600 mgof the corresponding peptide resin with H₂ O/triethylsilan/TFA.

Mass spectral analysis of the crude polypeptide indicated the presenceof the species of interest with a Molecular Weight (MW) of 9301 amongother low MW components (FIG. 1).

Ninety mg of crude polypeptide were purified by immobilized metal ionaffinity chromatography (IMAC) on a 25 ml volume Ni-NTA agarose column(FIG. 2). After desalting by size exclusion liquid chromatography 35 mgof "His-tag PbCS 242-310" were obtained. The measurement of theabsorbance at 280 nm of the eluted material (35 mg) and of the flowthrough (55 mg) indicated that the yield of the purification protocolwas 100%.

The Ni-column purified material was then cleaved with CNBr to eliminatethe 6xHis tag.

The digested material was reloaded on the Ni-column to eliminate theuncleaved peptide and treated with 10% mercaptoethanol at pH 8.0 inorder to reduce the methionine sulfoxide inserted during the synthesisand the Cys residue protective groups. The complete reduction of themethionine sulfoxide was confirmed by retreating the material with CNBrand checking the efficacy of the cleavage by mass spectrometry.

Further purification by size exclusion chromatography resulted in 19 mgof purified PbCS 242-310.

In FIG. 3 the mass spectrum of the obtained material is shown and inFIG. 4 the analytical chromatographic profiles of crude and purifiedpeptide are compared. The difference of retention times between the tworuns is due to the absence of the highly charged His tag in the purifiedmaterial. The purified "PbCS 242-310" was found to be about 95% purebased on the integration of peak areas when analyzed at 214 nm. Theamino acid composition of the CS polypeptide was consistent with thatexpected for this peptide (Table 1).

                  TABLE 1                                                         ______________________________________                                        Amino acid analysis                                                                     Residues per mole of PbCS 242-310                                   Amino acid.sup.a                                                                        Expected      Observed.sup.b                                                                          S.D..sup.c                                  ______________________________________                                        Asp + Asn 10            9.4       1.0                                           Glu + Gln 9 9.1 0.9                                                           Ser 7 8.4 2.1                                                                 Thr 4 2.2 0.8                                                                 Gly 3 3.1 1.0                                                                 Ala 2 3.4 1.1                                                                 Arg 4 4.8 0.9                                                                 Pro 1 1.1 0.2                                                                 Val 3 2.9 0.3                                                                 Met 1 1.0 0.1                                                                 Ilc 7 6.3 0.7                                                                 Leu 3 3.3 0.2                                                                 Phe 1 1.1 0.1                                                                 Lys 8 6.7 1.0                                                                 His 0 -- --                                                                   Tyr 1 1.2 0.2                                                               ______________________________________                                         .sup.a Both Cys and Trp are not determinated                                  .sup.b Mean value of five determination                                       .sup.c Standard deviation                                                

3. Discussion

Chemical synthesis of bioactive peptides has become a widespread andrapidly growing technique due to the automation and efficient protocolsfor chain assembly. For most applications, the crude synthetic productmust be purified to remove residuals reactants, failure sequences andchemically modified peptide species. This is usually accomplished byreversed-phase HPLC using aqueous trifluoroacetic acid/acetonitrilemobile phases. Although peptide synthesis has become highly automated,purification is still largely a manual process and thereforetime-consuming, expensive and not very efficient.

This example has shown that the process of the invention leads to highpurity as follows from FIG. 3 and is easy to perform and universallyapplicable.

The problem of a specific cleavage due to the presence of Met residuesin the desired sequence, as in the case described herein, is accordingto the invention easily circumvented using Met-sulfoxide residues whichare resistant to CNBr treatment and quantitatively reducible withmercaptoacetic acid¹⁴.

From the above it follows that the process of the invention wassuccessfully applied for the purification of the polypeptide "PbCS242-310", a 69 residue chain corresponding to C-terminal region of P.berghei CS protein¹². Although many peptide impurities were present inthe crude material after cleavage from the resin as shown by massspectral analysis reported in FIG. 1, the inventors were able to purifythe target peptide to homogeneity in high yield and in relatively shorttime. The complete protocol of purification yielded 19 mg of purifiedPbCS 242-310, corresponding to about 20% of the crude material.

In conclusion, it was demonstrated here that CNBr cleavage andprotection of relevant Met residues as sulfoxides, coupled with anefficient affinity tag can represent an efficient and general tool forthe purification of chemically synthesized long chain peptides.

REFERENCES

1. Chong, P., Sia, C., Tam, E., Kandil, A. & Klein, M. InternationalJournal of Peptide & Protein Research 41, 21-27 (1993).

2. Haaheim, L. R., Maskell, J. P., Mascagni, P. & Coates, A. R.Scandinavian Journal of Immunology 34, 341-350 (1991).

3. Roggero, M. A., et al. Molecular Immunology 32, 1301-1309 (1995).

4. Smith, D. D., et al. International Journal of Peptide & ProteinResearch 44, 183-191 (1994).

5. Lobl, T. J., Deibel, M. J. & Yem, A. W. Analytical Biochemistry 170,502-511 (1988).

6. Tomasselli, A. G., et al. Journal of Biological Chemistry 267,10232-10237 (1992).

7. Ball, H. L. & Mascagni, P. International Journal of Peptide & ProteinResearch 40, 370-379 (1992).

8. Krieger, D. E., Erickson, B. W. & Merrifield, R. B. Proceedings ofthe National Academy of Sciences of the United States of America 73,3160-3164 (1976).

9. Lindeberg, G., Tengborn, J., Bennich, H. & Ragnarsson, U. J.Chromatography 156, 366-369 (1978).

10. Porath, J., Carlsson, J., Olsson, I. & Belfrage, G. Nature 258,598-599 (1975).

11. Lindeberg, G., Bennich, H. & Engstrom, A. International Journal ofPeptide & Protein Research 38, 253-259 (1991).

12. Lanar, D. E. Mol. Biochem Parasitol. 39, 151-154 (1990).

13. Knecht, R. & Chang, J. Y. Analyt. Chem. 58, 2375-2379 (1986).

14. Houghten, R. A. & Li, C. H. Methods in enzymology 91, 549-559(1991).

15. Barany, G. & Merrifield, R. B. Peptides, analysis, synthesis,biology 1-163-165 (Academic Press, London, 1980).

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 1                                        - - <210> SEQ ID NO 1                                                        <211> LENGTH: 9                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: His tag sequence                                      - - <400> SEQUENCE: 1                                                         - - His His His His His His Gly Gly Met                                       1               5                                                          __________________________________________________________________________

What is claimed is:
 1. A method for the modification of (poly)peptidesfor facilitating purification thereof, comprising the steps of:insertingat least one specifically cleavable methionine and a tag at the end of a(poly)peptide chain during synthesis thereof and protecting methionineresidues within the (poly)peptide, if present, against cleavage, byprotecting them with a sulfoxide group, wherein the tag is one of a histag and a large molecule.
 2. A process for the preparation of purified(poly)peptides using the modification method of claim 1, comprising thesteps of:a) synthesizing the desired (poly)peptide; b) adding at leastone specifically cleavable methionine at the end of the (poly)peptide,while having protected methionine(s) within the (poly)peptide, ifpresent, against cleavage by protection with a sulfoxide group; c)elongating the (poly)peptide and the methionine(s) added thereto with atag to obtain an elongated (poly)peptide; d) purifying the elongated(poly)peptide by means of a tag-specific purification method; and e)removing the tag and the additional methionine(s) from the elongated(poly)peptide by means of a cleavage method based on cyanogen bromide toobtain purified (poly)peptide, wherein the tag is one of a his tagsequence and a large molecule.
 3. The process as claimed in claim 2,wherein the his tag sequence consists ofHis-His-His-His-His-His-Gly-Gly-(SEQ ID NO: 1).
 4. The process asclaimed in claim 2, wherein the tag-specific purification method ischromatography based on affinity for the tag sequence.
 5. The process asclaimed in claim 2, wherein the tag is a large molecule and thetag-specific purification method is gel filtration chromatography. 6.The process as claimed in claim 2, wherein the desired (poly)peptide isproduced by recombinant DNA techniques in a living host and furthercomprising the step of deleting or substituting with another amino acidany methionines within the (poly)peptide.
 7. The process as claimed inclaim 6, wherein the living host is one of a eukaryotic host and aprokaryotic host.
 8. The process as claimed in claim 5, wherein the tagis polyethylene glycol.